Tethering Growth Factors to Collagen Surfaces Using Copper-Free

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Tethering Growth Factors to Collagen Surfaces Using Copper-free Click Chemistry: Surface Characterization and In Vitro Biological Response Hyun Jong Lee, Gabriella Fernandes-Cunha, Ilham Putra, Won-Gun Koh, and David Myung ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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ACS Applied Materials & Interfaces

Tethering Growth Factors to Collagen Surfaces Using Copper-free Click Chemistry: Surface Characterization and In Vitro Biological Response Hyun Jong Lee, †, ‡ Gabriella M. Fernandes-Cunha, †, ‡ Ilham Putra, § Won-Gun Koh, *, and David Myung *, ‡, ⊥

‡

Byers Eye Institute at Stanford, Stanford University, Palo Alto, CA 94303, USA

§

Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of

Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. ⫽

Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro,

Seodaemun-gu, Seoul 03722, Republic of Korea ⊥

VA Palo Alto Health Care System, Palo Alto, CA 94304, USA

⫽ KEYWORDS Wound healing; Direct growth factor surface-coupling; Epidermal growth factor (EGF); Copperfree click chemistry; Strain-promoted azide-alkyne cycloaddition (SPAAC); Biocompatible chemical reaction

1 ACS Paragon Plus Environment


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◼ ABSTRACT

Surface modifications with tethered growth factors have mainly been applied to synthetic polymeric biomaterials in well-controlled, acellular settings, followed by seeding with cells. The known bio-orthogonality of copper-free click chemistry provides an opportunity to not only use it in vitro to create scaffolds or pro-migratory tracks in the presence of living cells, but also potentially apply it to living tissues directly as a coupling modality in situ. In this study, we studied the chemical coupling of growth factors to collagen using biocompatible copper-free click chemistry and its effect on the enhancement of growth factor activity in vitro. We verified the characteristics of modified epidermal growth factor (EGF) using mass spectrometry and EGF/EGF receptor binding assay, and chemical immobilization of EGF on collagen was also evaluated by copper-free click chemistry using surface x-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR) spectroscopy, and enzyme-linked immunosorbent assay (ELISA). We found that the anchoring was non-cytotoxic, biocompatible, and sufficiently rapid for clinical application. Moreover, the surface-immobilized EGF has significant effects on epithelial cell attachment and proliferation. Our results demonstrate the possibility of copper-free click chemistry as a tool for covalent bonding of growth factors to extracellular matrix collagen and the potential effectiveness of immobilized EGF in this setting. This approach is a novel and potentially clinically useful application of copper-free click chemistry as a way of directly anchoring growth factors to collagen and foster epithelial wound healing.


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◼ TABLE OF CONTENTS / ABSTRACT GRAPHIC

◼ INTRODUCTION Growth factors are well-studied candidate therapeutics for improving wound healing.1-2 Growth factors have important roles in wound healing as well, not only as a direct stimulator of epithelialization, but also as a signal for recruiting stem cells.3 A growth factor-based therapeutic that reduces healing time with few administrations would be of great benefit for treating injured, poorly healing tissues.1-3 In clinical trials, the successful regenerative effects of growth factors have been limited.4-6 One approach to improving the therapeutic potential of growth factors is surface-coupling, in which covalently immobilized growth factor exerts prolonged effects than in soluble form, presumably by increasing resistance clearance by endocytosis and other mechanisms.7-8 Chemical immobilization of growth factors has been widely studied on various surfaces through photochemical methods as well as water-soluble carbodiimide for tissue repair and regeneration.7-8 Immobilized growth factors are not only more resistant to endocytosis, but also have enhanced effects by multivalency and inhibition of down-regulation.7 N-hydroxysuccinimide (NHS) ester are commonly used for coupling between amine group. 9-11 The NHS ester reaction occurs in mild conditions for proteins or peptides which have primary amines available for the modification at the N-terminus and in the lysine.12 Based on this



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principle, there have been some trials to use homo-bifunctional NHS ester to attach growth factors on amine coated surfaces, and they showed favorable immobilization efficiency in wellcontrolled environments.10-11 However, the NHS ester group reacts with biomolecules that have primary amine groups, so homo-bifunctional NHS ester would be highly problematic in vivo environments. To avoid undesirable reaction, the tissue-growth factor coupling system should include high reaction selectivity as well as simplicity for direct using chemical reaction to tissues. Click chemistry is a water-friendly, biocompatible and highly selective reaction, so it has been extensively used for biomolecular tagging and labeling in pharmacological and biomedical fields.13-15 Strain-promoted azide-alkyne cycloaddition (SPAAC) is a type form of click chemistry that is particularly useful for biological systems through its bioorthogonality—it forms a triazole ring bond through [3+2] cycloaddition of azide and cyclooctyne without any external catalysts or side products. Here, we have chemically immobilized growth factor onto collagen surface by SPAAC and verified it using various surface analysis. We chose epidermal growth factor (EGF) which is a prototypical trophic factor for epithelial cells,3 and it also enhances wound healing.1,

3

We

modified the EGF via NHS ester chemistry to conjugate azide functional group for SPAAC, and the properties of azide-conjugated EGF were evaluated. The cyclooctyne-modified collagen surface generated via NHS ester chemistry was applied for damaged model tissue, and we immobilized the modified growth factor to the surface through the SPAAC reaction. Qualitative and quantitative analyses were performed to prove covalent bonding of growth factors, and the biological effect of chemically immobilized EGF was evaluated in vitro.


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◼ EXPERIMENTAL SECTION Materials. Unless otherwise noted, all chemicals and solvents were of analytical grade and used as provided form by the manufacturers. Dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO-sulfo-NHS ester), Tween-20, gelatin, collagenase, fibronectin, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), Cholera Subunit A, insulin, cell counting kit-8 and Triton-X were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) pH 7.4, Slide-A-Lyzer™ dialysis cassette kit (3.5k MWCO), collagen I bovine protein, epidermal growth factor recombinant human protein (EGF) and EGF human enzyme-linked immunosorbent assay (ELISA) kit (30X wash buffer, biotinylated antibody reagent, streptavidinHRP concentrate, TMB substrate and stop solution containing 0.16M sulfuric acid), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) with 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), fetal bovine serum (FBS), Insulin-Transferrin-Selenium (ITS), Dulbecco's phosphate-buffered saline (DPBS), antibiotic-antimycotic, keratinocyte-serum free media (KSFM), bovine pituitary extract (BPE), LIVE/DEAD Viability/Cytotoxicity kit, trypsin-EDTA, paraformaldehyde, Alexa Fluor Phalloidin 647 and 4',6-Diamidino-2Phenylindole (DAPI) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Azido-poly(ethylene glycol)5-sulfo-N-hydroxysuccinimidyl ester (Azide-PEG-sulfo-NHS ester) was purchased from BroadPharm (San Diego, CA, USA). AlphaLISA EGF/EGFR Binding kit (Anti-Human IgG Fc-specific AlphaLISA acceptor beads, streptavidin-coated donor beads, biotinylated EGF, EGFR-Fc and AlphaLISA immunoassay buffer) was purchased from PerkinElmer (Waltham, MA, USA). Sensor chip CM5, HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) and amine coupling kit (1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 1.0 M



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ethanolamine-HCl pH 8.5) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA).

EGF conjugation and characterization. EGF was prepared as 1.0 mg/mL (0.16 mM) solution in PBS. Azide-PEG-sulfo-NHS ester and DBCO-sulfo-NHS ester were dissolved in PBS as 100 mg/mL (0.20 M and 0.19 M, respectively) before mixing. 100 µl of 1.0 mg/mL EGF solution were mixed with 4.1 µL of 100 mg/mL azide-PEG-sulfo-NHS ester or 4.2 µL of 100 mg/mL DBCO-sulfo-NHS ester solution (50-fold molar excess of EGF), and the reaction was allowed to proceed for 2 hours at 4 °C. To remove unconjugated azide-PEG-sulfo-NHS ester and DBCO-sulfo-NHS ester, the mixed solution was dialyzed using dialysis cassette kit overnight at 4 °C. The resultant azide-PEG-conjugated EGF (Azide-PEG-EGF) and DBCO-conjugated EGF (DBCO-EGF) were analyzed by matrix-assisted laser desorption and ionization time of flight (MALDI-TOF) mass spectrometry (Perspective Voyager-DE RP Biospectrometry instrument, Framingham, MA, USA) at the Stanford Protein and Nucleic Acid Biotechnology Facility (Stanford, CA, USA). To evaluate bioactivity of conjugated EGF, we followed the instruction provided with AlphaLISA EGF/EGFR Binding kit for measuring bioactivity of Azide-PEG-EGF and DBCOEGF. Anti-human IgG Fc-specific AlphaLISA acceptor beads and EGFR-Fc were mixed according to the instruction and incubated for 30 minutes at room temperature. The mixture solution was added to the each well that has 1.6125 – 100 µg/mL of EGF, Azide-PEG-EGF or DBCO-EGF with biotinylated EGF, and then it was incubated for 30 minutes at room temperature. Streptavidin-coated donor beads were added to the each well and incubated for 30


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minutes at room temperature, and then the AlphaLISA optical signal of resultant each well was measured at 615 nm using AlphaLISA mode of TECAN M1000 Pro (San Jose, CA, USA) at the Stanford High-Throughput Bioscience Center (Stanford, CA, USA).

EGF coupling on collagen surface and characterization. A Glass slide (VWR, Radnor, PA, USA) was cut into 1 × 1 cm2. To coat collagen on glass substrates, 0.1 mg/mL collagen I bovine protein solution was dropped and incubated overnight at 4 °C. The collagen coated substrate was washed and dried gently. On the collagen surface, 0.1 mg/mL DBCO-sulfo-NHS ester solution in PBS was applied and incubated for 30 minutes at room temperature to introduce DBCO group onto the surface. The resultant surface was washed and dried gently. For the copper-free click reaction, 0.01 mg/mL Azide-PEG-EGF was applied on the DBCO-modified surface and incubated 60 minutes at room temperature and then washed. For the physical adsorption sample, 0.01 mg/mL EGF was used onto the collagen surface and incubated for 60 minutes at room temperature and then washed without DBCO-sulfo-NHS ester reaction step. Tween-20 was used for washing solution and repeated five times at each washing step. An ellipsometry (Jobin-Yvon UVISEL spectroscopic ellipsometry, Horiba Scientific, Edison, NJ, USA) was used to measure surface thickness. The investigated wavelength was 200 to 850 nm, and the angle was fixed at 70 °. The fitting of the experimental data was performed using DeltaPsi2 software (Horiba Scientific, Edison, NJ, USA). Thermo K-Alpha X-ray photoelectron spectroscopy (XPS) spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure elemental compositions with monochromatic AlKα X-ray source (1486.6 eV, 36 W, sampling area; 400 µm diameter). Survey



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spectra were collected using a pass energy of 200 eV with a step size of 1.0 eV, while highresolution spectra were collected at a pass energy of 40 eV with a step size of 0.1 eV.

Measurement of EGF immobilization using surface plasmon resonance (SPR). A Biacore X100 (GE Healthcare Life Science, Pittsburgh, PA, USA) was used for observing the EGF immobilization on the collagen surface in real time. HBS-EP buffer was used as running buffer. On a CM5 chip, EDC/NHS of amine coupling kit was treated to make NHS active surface, and then 0.1 mg/mL collagen I bovine protein solution was injected. To deactivate unreacted NHS, 1.0 M ethanolamine-HCl was injected after collagen immobilization. 0.1 mg/mL DBCO-sulfoNHS ester was injected to conjugate DBCO group on to the collagen surface, and 1.0 M ethanolamine-HCL was injected again to deactivate NHS ester group of the unreacted DBCOsulfo-NHS ester. 0.1 mg/mL Azide-PEG-EGF was injected onto the DBCO-conjugated surface, and the running buffer was flowed to observe dissociation of immobilized EGF. To compare physical adsorption of EGF, 0.1 mg/mL EGF was injected after deactivation following collagen coating without DBCO-sulfo-NHS ester and second deactivation step. The response curve and values were obtained by subtraction of reference surface signal and analyzed using Biacore X100 evaluation software (GE Healthcare Life Science, Pittsburgh, PA, USA).

Quantification of immobilized EGF using enzyme-linked immunosorbent assay (ELISA). We followed the EGF ELISA kit protocol with some minor modifications. Briefly, before the ELISA procedure, the EGF was immobilized onto the surface. 0.1 mg/mL collagen I bovine protein solution was filled in 96 well plate and incubated overnight at 4 °C to coat collagen on the surface of the well plate, and 0.1 mg/mL DBCO-sulfo-NHS ester solution was added to wells


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and incubated for 30 minutes at room temperature. To chemically couple EGF by copper-free click chemistry, 0.001 mg/mL Azide-PEG-EGF was added to wells and incubated for 10, 30 and 60 minutes at room temperature. For physical adsorption, 0.001 mg/mL EGF was added after collagen coating for 60 minutes at room temperature. To begin ELISA procedure, biotinylated antibody reagent and diluted streptavidin-HRP in PBS were added to the well and incubated for 2 and 1 hours in turn, respectively. After each step including EGF immobilization and ELISA, all wells were washed three times using the washing buffer in the kit. For the color development, TMB solution was added and incubated in the dark for 30 minutes at room temperature, and stop solution was added to stop the reaction without washing. The absorbance of resultant each well was measured at 550 nm using SpectraMax M Series Multi-Mode Microplate Reader (Sunnyvale, CA, USA). For quantification of immobilized EGF, a standard curve of EGF ELISA was obtained following the established protocol of instruction, and the amounts of EGF were calculated.

Cell isolation and culture. Primary corneal keratocytes were obtained from rabbit's corneas. First, plates were pre-coated with gelatin and DI water (1:1). After the sterilization of the eyes, the cornea was cut and the epithelium layer was mechanically removed. The cornea was cut into pieces and digest with 0.25 % collagenase at 37 °C for 2 hours. The stroma pieces were placed in the gelatin pre-coated plates and teased apart with forceps. After that, 1 mL of medium DMEM/F12 with HEPES containing FBS (10 %) and ITS (0.5 %) and incubated overnight. In the next day, the matrix debris were separated by a plastic cell sieve (20 µm pores). The suspension was centrifuged and the cells were resuspended in DMEM/F12 with HEPES and ITS. After confluence, the cells were subcultured and used at passage four.



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Primary corneal epithelial cells were obtained from rabbit's corneas. First, plates were precoated with a solution of collagen and fibronectin (1:1) and BSA (1 %) for 1 hour at 37 °C. Rabbit's eyes were then sterilized in 10 % betadine (Valley Vet Supply, Marysville, KS, USA) and washed with DPBS, in the presence of antibiotic-antimycotic. The corneas were placed side up on sterile surface and cut into triangular shaped wedges. Then, the segments were placed upside down on the pre-coated plates. The tissue was allowed to dry for 20 minutes, and then one drop of DMEM/F12 with HEPES, FBS (15 %), DMSO (0.5 %), Cholera Subunit A (1 µg/mL), EGF (10 ng/mL) and insulin (5 µg/mL) was added to each the segment. On the next day, 1 mL of medium was added to each well. On day 5, the segments were removed and the medium was changed to KSFM containing BPE and EGF. After confluence, the cells were subcultured and used at passage four.

In vitro stromal cell viability assay. To evaluate the cytotoxicity of DBCO-sulfo-NHS ester and Azide-PEG-EGF, primary keratocytes were seeded on polystyrene 48 well plates in a concentration of 5 × 104 cells/mL and incubated overnight. In the next day, each 0.00625 – 0.1 mg/mL of DBCO-sulfo-NHS ester and Azide-PEG-EGF in DPBS was added to the cells. After 15 minutes, cell viability was assessed via LIVE/DEAD staining following the manufacturer’s instructions. The plates were mounted and observed using laser scanning microscope (ZEISS LSM 880, Carl Zeiss Ag, Oberkochen, Germany). The numbers of viable and dead cells were analyzed using image J software (National Institutes of Health, Bethesda, MD, USA). Cell viability was obtained by diving the number of live cells to number of total cells. To confirm cell viability in optimal condition of SPAAC reaction, polystyrene 48 well plates were coated with collagen solution for 30 minutes at 37 °C. Then, primary keratocytes were


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seeded on polystyrene 48 well plates in a concentration of 5 × 104 cells/mL and incubated overnight. In the next day, 0.025 mg/mL of DBCO-sulfo-NHS ester in DPBS was added to the wells and placed at 37 °C. After 15 minutes, the wells were gently washed using DPBS three times, and 0.01 mg/mL of Azide-PEG-EGF in serum free medium was added. The plate was placed at 37 °C for 1 hour and gently washed using DPBS three times, and the cells were stained via LIVE/DEAD staining. The cells were observed using laser scanning microscope.

In vitro corneal epithelial cell proliferation assay and immunostaining. Ninety-six well plates were coated with 0.1 mg/mL of collagen solution for 30 minutes at 37 °C. Then, 50 µL of DBCO-sulfo-NHS ester solution (0.025 mg/mL in DPBS) was applied to the collagen coated 96 well plates and placed at 37 °C for 15 minutes. After gentle washing using DPBS three times, 50 µL of Azide-PEG-EGF solution (0.01 mg/mL in DPBS) was added and the plate was placed at 37 °C for 1 hour and gently washed using DPBS three times. Prior to the seeding, medium was substitute for KSFM medium without EGF and BPE and the cells were incubated overnight. In the next day, 4 mL of trypsin (0.25 %) was added to the cells for 2 minutes. The trypsin-EDTA was neutralized by the addition of 6 mL of medium containing serum. The cells were centrifuged at 12,000 rpm for 5 minutes. The medium was aspirated and the cells were resuspended in KSFM medium without BPE and EGF. The cells were plated on the SPAAC-treated surfaces at a density of 1,000 cells per well, and KSFM medium without BPE and EGF was added to each well. For the EGF-free group, KSFM without BPE and EGF was added to each well after seeding cells to the collagen-coated wells. For the soluble EGF group, KSFM medium containing 0.01 mg/mL of EGF was used after seeding cells to the collagen-coated wells. Cell proliferation was accessed using cell counting kit-8 following the manufacturer’s instructions.



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For immunostaining, glass plates were coated with collagen at 37 °C for 30 minutes. Then the SPAAC treatment was performed as described above. After the reaction was completed, DPBS solution containing 1 % BSA was added for 1 hour at room temperature. For the EGF-free and soluble EGF group, 1 % BSA was added to the collagen-coated wells for 1 hour at room temperature. Then the wells were washed three times with DPBS. Corneal epithelial cells were added to the treated wells following the same procedure described above. At 24 hours after seeding, the wells were washed with DPBS and fixed with 4 % paraformaldehyde for 15 minutes. After washing with DPBS, the cells were permeabilized for 30 minutes with 1 % BSA and 0.1 % Triton-X. Next, Alexa Fluor Phalloidin 647 was added and incubated for 20 minutes. After washing three times with DPBS, 5 mg/mL of DAPI solution in DPBS was added and incubated for 5 minutes. The glass plates were mounted and the morphologies were observed using fluorescence microscopy. To quantify the cells area, image J software was used.

Statistical analysis. The bar graphs are represented as mean ± standard deviation. Statistical difference between samples was analyzed by one-way ANOVA and two-way ANOVA. For all statistical tests, a threshold value of p = 0.05 was chosen. ns indicates not significance, and 0.01 < p < 0.05 (*) and p < 0.01 (**) indicate difference.

◼ RESULTS Overall procedure for epidermal growth factor coupling by click chemistry. Collagen is a good model substrate to mimic damaged tissue surface. The material is more than 30 % of total protein and distributed in many tissues such as skin, tendon, cornea and bone.16-17 Thus, when tissues are injured, there is a high probability that collagen is exposed.


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Our strategy for chemical immobilization of epidermal growth factor (EGF) onto collagen was copper-free click chemistry, specifically strain-promoted azide-alkyne cycloaddition (SPAAC), which makes it possible to have high reaction efficiency without interruptions of many kinds of biomolecules in body fluid.18 Moreover, since SPAAC process is considerably simple, we could use the commercial reagents without additional synthesis or modification of chemicals. A significant fact on this reaction is that we used only water and pH 7.4 phosphate-buffered saline (PBS) as solvents in the entire reaction process, and it means that our proposed reaction has the potential to be applied as a clinical therapy. Figure 1 shows the overall schematic of chemical immobilization of EGF. The process consists of two parts that begins with dibenzocyclooctyne (DBCO) group tethering to the surface and the combination of azide-conjugated EGF with the DBCO group by SPAAC. The Nhydroxysuccinimide (NHS) ester reaction was applied in order to introduce SPAAC moieties to the collagen and EGF. The NHS ester group of DBCO-sulfo-NHS ester and azide-poly(ethylene glycol)5-sulfo-NHS ester (Azide-PEG-sulfo-NHS ester) reacted with primary amine groups of collagen and EGF, respectively. The sulfo-NHS ester group has an identical role to the NHS ester group, but sulfo-NHS ester is more useful due to its water-solubility. The NHS ester group itself is highly reactive and unstable, however, once the DBCO or azide group is conjugated to the biomolecule, the SPAAC moieties have long-term stability.19 When the azide group on the EGF comes in contact with the DBCO groups tethered on the surface, the SPAAC reaction occurs and generates stable triazole bonding, leading to covalent immobilization of EGF onto the collagen surface.



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Figure 1. Schematic image of EGF coupling on the collagen surface by copper-free click chemistry.

Conjugation of click chemistry moiety on epidermal growth factor. The NHS ester-based Azide-PEG-sulfo-NHS ester and DBCO-sulfo-NHS ester were used to conjugate click chemistry moieties onto growth factors without additional chemical modification. The simple coupling reaction by mixing NHS ester and EGF yielded azide- and DBCO-conjugated EGF for click chemistry with incubation at 4 ˚C for 2 hours. The molecular weight differences by reaction of EGF with Azide-PEG-sulfo-NHS ester were measured by MALDI-TOF (Figure 2a). The molecular weight per charge of unmodified EGF was 6208.65 Da, and the Azide-PEG-conjugated EGF (Azide-PEG-EGF) showed the sharp peaks at 6525.34, 6841.42, and 7159.56 Da. The increased degrees were 316.69, 632.77, and 950.91 Da, and these are consistent with the molecular weight of Azide-PEG group. Thus, the 6525.34, 6841.42, and 7159.56 Da represent that the EGF has one, two and three Azide-PEG


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group, respectively. A human EGF has three NHS ester reactive sites in the N-terminal and side chain of two lysines (Lys28, Lys48), and the N-terminal amine has higher nucleophilicity than lysine.10,

20

We could quantify approximate relative ratio via MALDI-TOF peak areas. The

conversion from EGF to Azide-PEG-EGF was about 90 %, and the ratio of the EGF has one, two and three Azide-PEG group was about 2 : 2: 1. The spectra of DBCO-conjugated EGF (DBCOEGF) showed a broad peak from 6200 to 7500 Da. While the reasons for this are not clear, we determined that DBCO conjugation to EGF is problematic in some way, possibly due to hydrophobic interactions between DBCO and EGF.

Figure 2. (a) MALDI-TOF spectra and (b) inhibition curve on EGF/EGFR binding of EGF, Azide-PEG-EGF and DBCO-EGF.

Since EGF exerts their effects on cells by binding to the EGF receptor (EGFR) on cell membranes, the bioactivity of EGF was verified by binding with EGFR. So, the binding affinity of Azide-PEG-EGF and DBCO-EGF with EGFR was estimated by its ability to inhibit AlphaLISA signaling. Briefly, the assay system we used is based on the singlet oxygen formation from donor bead which was coated with streptavidin, and transfer in limited distance



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to the acceptor bead which was bound with EGFR, and light emission from acceptor bead by singlet oxygen.21 If a biotinylated EGF was added to the system, the biotin and EGF parts combined with streptavidin and EGFR, respectively. From the specific couplings, the donor and acceptor bead keep the close distance to make acceptor bead emits light.21 In the system, if bioactive and non-biotinylated EGF are applied together with biotinylated EGF, a competitive reaction occurs between non- and biotinylated EGF with EGFR, and the light emission becomes reduced. We measured the emission from acceptor bead after mixing biotinylated EGF with EGF, Azide-PEG-EGF and DBCO-EGF. As we expected, EGF showed a strong inhibition effect. At a concentration above 6.25 µg/mL, EGF totally suppressed the binding assay signal, and the signals were gradually increased with decreasing concentration of EGF. The Azide-PEGEGF also showed an inhibition, but not as much as EGF. When we calculate the inhibition efficiency of Azide-PEG-EGF using linear interpolation at the points 25, 12.5 and 6.25 µg/mL of Azide-PEG-EGF, Azide-PEG-EGF showed 42.18 ± 7.08 % of inhibition capacity compared to EGF. On the other hand, DBCO-EGF did not affect the signal change, which means that there was no competitive reaction between DBCO-EGF and biotinylated EGF. DBCO-EGF was not bioactive, so it did not bind to EGFR. We then proceeded with subsequent experiments using only the azide-PEG-EGF.

Collagen surface-coupling of epidermal growth factor. The Azide-PEG-EGF was reacted with DBCO groups on collagen surfaces. To predict the thickness variation after surface modification, the thicknesses of surfaces were measured using ellipsometry, equipment that measures surface thickness optically (Figure 3). Generally crystalline materials that have fixed refractive index are good for ellipsometry. However, in the case of proteins and peptides


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including collagen and EGF in our system, proper multi-layer refractive index information and modeling of several steps are required.22 Thus, here, we aimed to compare relative thickness through rough modeling with a reference assuming the multi-layer as a homogenous layer.23-24 The collagen coating on the glass substrate thickness was detectable, and the estimated thickness of collagen coating was 3.60 ± 0.24 nm. To compare the immobilization between physical adsorption and chemical bonding, the EGF solution was incubated on the collagen surface for 30 minutes. After gently washing with 0.05 % Tween-20 solution five times, the resultant estimated thickness was 3.51 ± 0.01 nm. The protein layer thickness was maintained, meaning that the EGF was not bound on the surface because the EGF was not strongly attached. When the AzidePEG-EGF was applied on the surface after DBCO group tethering on collagen surface, the estimated thickness was increased to 4.61 ± 0.48 nm. From this result, there was a significant difference between physical and chemical immobilization of EGF, and it showed that the anchored EGF by copper-free click chemistry has stronger than physical adsorption.

Figure 3. Surface thickness change on the substrate after treatment measured by ellipsometry.



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As we mentioned, the ellipsometry result assures only relative comparison of surface thicknesses, so the samples were also measured using X-ray photoelectron spectroscopy (XPS) to obtain data in elemental compositions (Figure 4). In general, elemental composition analysis of XPS is performed 10 nm from the top surface. From this principle, we could estimate the thickness of layers on the substrate by the Si peak, because it decreases upon addition of protein to the surface. The collagen-coated surface showed reduced peaks relevant to the Si element by the deposition of collagen. Nevertheless, the Si peak was evident, which infers that the thickness of the collagen coating was under 10 nm, and the physical adsorption of EGF had almost similar thickness to collagen coating with the overlapped spectrum. For the substrate immobilized with EGF by SPAAC, the Si spectrum was significantly reduced, which represents increasing thickness and strong bonding between EGF and collagen. The result of Si elemental spectra corresponds with thickness change in ellipsometry measurement, which shows that the coupling by SPAAC leads to chemical immobilization of Azide-PEG-EGF on the collagen surface.


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Figure 4. XPS spectra of (a) overall, (b) Si, (c) C 1s and (d) N 1s.

The protein thickness change was verified by decreasing Si elemental composition, but C=O peak did not increase significantly despite immobilization of EGF which has peptide bonds when the EGF was immobilized by adsorption and SPAAC. This result means that the addition of EGF on the collagen surface leads to few differences in the carbon elemental composition. This is because the total amount of EGF is much lower than collagen. On the other hand, the height of the C-C peak was significantly increased by SPAAC, which suggests a high density of DBCO groups on the collagen surface.



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When the collagen or EGF were immobilized on the surface, there were significant rises in N 1s peaks. However, the differences between the SPAAC and collagen coating in height were lower relative to the Si or C 1s peaks. As we mentioned in the C 1s spectra, immobilization of EGF had no big effect on elemental composition, moreover the primary amines of EGF and collagen were consumed for click chemistry. Therefore, SPAAC showed similar N 1s height, even though it contained more proteins. Surface plasmon resonance (SPR) was used to monitor the real-time kinetics of EGF immobilization on the surfaces (Figure 5). The collagen was chemically immobilized on the SPR chip first by EDC/NHS chemistry, then the EGF was injected into the physical adsorption channel, and then the Azide-PEG-EGF was applied after DBCO-sulfo-NHS ester reaction within the SPAAC channel. The final variation by physical adsorption and SPAAC were 85.5 and 704.8 RU, respectively. EGF coupling by SPAAC exhibited a sharp increase for about 300 seconds, and then transitioned to a slower rate (Figure 5). The decreased RUs by dissociation in SPAAC and physical adsorption were 69.1 and 93.1 respectively, which was derived by physically adsorbed EGFs in both cases. Although the amount of detached EGF was higher in SPAAC than physical adsorption, it was a relatively small proportion of the total amount attached chemically. These results indicated chemical immobilization of EGF to the surface by reaction of the pendant azide group on EGF and the pendant DBCO group on the surface collagen.


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Figure 5. SPR data for the immobilization EGFs by SPAAC and physical adsorption.

To confirm the effect of concentration and reaction time on immobilization, we measured the amount of surface-immobilized growth factors quantitatively by enzyme-linked immunosorbent assay (ELISA) (Figure 6). To evaluate the EGF immobilization depending on the concentration of DBCO-sulfo-NHS ester, we applied 0.001 – 1 mg/mL of DBCO-sulfo-NHS for collagen surface modification. After the surface modification, 0.001 mg/mL of Azide-PEG-EGF was used for EGF immobilization by SPAAC. Because the detectable linear range of EGF ELISA was 80 pg/cm2, we could not obtain the accurate amounts due to saturation in the above region. Except for 1 mg/mL because of saturation, a notable increase of immobilized EGF was noticeable with increasing DBCO-sulfo-NHS ester concentration (Figure 6a). To observe the effect of AzidePEG-EGF concentration, collagen surfaces were treated with 0.1 mg/mL DBCO-sulfo-NHS ester. Azide-PEG-EGF also showed significant increase trend similar to DBCO-NHS-ester (Figure 6b). When native EGF was incubated for 30 minutes after collagen coating on the well plate, the residual amount of EGF by physical adsorption was 1.11 ± 0.11 pg/cm2, and this result suggests that there was hardly any EGF left on the surface. For the EGF coupling by click



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chemistry, we measured the amount of EGF left on the surface depending on the reaction time. After tethering the DBCO group onto the collagen surface, the Azide-PEG-EGF was applied and incubated for 10, 30, and 60 minutes. The amounts of immobilized EGF were 18.62 ± 0.28, 46.75 ± 0.57, and 69.76 ± 0.94 pg/cm2 for 10, 30, and 60 minutes of incubation times, respectively. The immobilized growth factors were increased with SPAAC reaction time, and even the 10 minutes of incubation time showed more chemically immobilized EGF than physical adsorption. Generally, since the ELISA procedure included multiple washing steps, the results suggest that the surface immobilized EGF by SPAAC is strongly bonded with the collagen surface.

Figure 6. Quantification of immobilized EGF on the surface by physical adsoprtion and SPAAC depending on (a) DBCO-sulfo-NHS ester concentration, (b) Azide-PEG-EGF concentration and


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(c) reaction time. ✝ indicates estimated value of the saturated ELISA signal by extrapolation. The statistical significances indicate between physical adsoprtion and SPAAC depending on reaction time. (d) Residual amounts of immobilized EGF on the surface by SPAAC. The statistical significances indicate between day 0 and each time point.

The durability of immobilized EGF was also measured by tracking the surface concentration of EGF on the surface over time (Figure 6d). The EGF was first chemically immobilized by SPAAC after 60 minutes of reaction time, and EGF on the surface gradually decreased with time. There was no significant difference after one day from reaction, while 75.52 ± 3.39% remained at day 4 on the surface. After 1 week, 75.84 ± 2.70% of EGF was maintained.

Biological response to immobilized epidermal growth factor in vitro. We evaluated the cytocompatibility on keratocytes depending on the concentration of Azide-PEG-EGF and DBCO-sulfo-NHS ester (Figure 7a). Azide-PEG-EGF maintained excellent cell viability in all concentrations under 0.1 mg/mL, representing that Azide-PEG modification of EGF does not impact the biocompatibility of the growth factor. The IC50 of DBCO-sulfo-NHS ester was about 0.05 mg/mL, and the cells maintained viability under about 0.025 mg/mL range. The viability result was considered by separating the effects of the DBCO and NHS ester group. In the case of DBCO group, it had no cytotoxicity and systemic toxicity under a concentration of 0.025 mg/mL.25 Also, the NHS ester reaction showed decreasing viability over 20 mg/mL concentration, which is also a higher level than reported by other groups.26 The combination of two chemical groups could give synergic effect to the cell viability, but the previous studies suggest that DBCO group is the main cause of low cell viability. DBCO-sulfo-NHS ester tended



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to decrease cell viability with increasing concentration beyond 0.025 mg/mL because the experiment was conducted at this concentration to find the maximum usable limit. Thus, 0.025 mg/mL of DBCO-sulfo-NHS ester with was used for subsequent SPAAC steps to tether EGF in further in vitro experiments.

Figure 7. (a) Effects of Azide-PEG-EGF and DBCO-sulfo-NHS ester on the viability of primary rabbit corneal keratocytes. Fluorescence images of primary rabbit corneal keratocytes LIVE/DEAD assay after (b) DBCO-sulfo-NHS ester treatment and (c) complete SPAAC reaction. Scale bars represent 250 µm.


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To confirm the biocompatibility of the SPAAC reaction itself, each step of the SPAAC reaction was applied to directly on cultured keratocytes. Keratocytes are a good model to predict biocompatibility, because they are dispersed throughout the stroma, and would be directly exposed to SPAAC reagents during application to a wounded tissue in vivo. Here, we used the 0.025 mg/mL concentration of DBCO-sulfo-NHS ester on the keratocytes. As we showed in Figure 7a, there was no cytotoxic effect on the DBCO-sulfo-NHS ester-treated cells (Figure 7b), and then Azide-PEG-EGF was applied to the DBCO-sulfo-NHS-treated cells. After completing SPAAC reaction on the keratocytes, the cells also exhibited good viability (Figure 7c). Therefore, our proposed chemical reaction is biocompatible enough to be directly applied to the keratocytes and potentially to the damaged tissues directly. To evaluate the effect of the chemically immobilized EGF by SPAAC, we observed the morphology and proliferation of corneal epithelial cells. It is hard to discern the effect of immobilized EGF in isolation because collagen is a cell adhesion protein, so the cells were cultured on the BSA-treated surface after collagen coating and SPAAC to compare the cell morphology. Twelve hours after cell seeding, nuclei and actin were stained. Regardless of the growth factor application, the epithelial cells showed similar slender and stretched shapes on the control surface and in the presence of soluble EGF (Figure 8a and b). On the other hand, cells were found to be widely spread into polygonal shapes with dense actin filament structures on the SPAAC surface (Figure 8c). Not only large cells but also small dense cells were also observed on the SPAAC surface, which suggests active cell division. The chemically immobilized EGF promotes the production of fibronectin, laminin, and integrin, which induces spread morphologies.27 When the cell areas were quantified, a significant difference between control and SPAAC surfaces was observed (Figure 8d). The average of cell area was not prominently



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high because of the cells of small size, but the relative cell area of SPAAC showed biased distribution toward larger cell area than control and soluble EGF.

Figure 8. Fluorescence images of actin (red) and nuclei (blue) stained corneal epithelial cells cultured (a) without EGF, (b) with soluble EGF, and (c) with chemically immobilized EGF by SPAAC. (d) Relative cell area per cell quantified based on the fluorescence images. The surfaces were treated by BSA after collagen coating and SPAAC to avoid adhesion effect of collagen. Scale bars represent 100 µm. (e) Relative cell proliferation of corneal epithelial cells cultured without EGF, with soluble EGF and with chemically immobilized EGF by SPAAC. The statistical significance is indicated between the control and other groups at each time point.


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Corneal epithelial cell proliferation assays showed results that were consistent with cell morphology (Figure 8e). The cells exposed to control media and soluble EGF did not show any significant differences, while the cells exposed to SPAAC showed increased proliferation over time. There was little difference between SPAAC and other groups at 24 hours, statistically significant differences were seen at 72 and 120 hours.

◼ DISCUSSION In this study, we report a novel application of copper-free click chemistry directed toward the coupling of growth factors onto collagenous surfaces. If our goal was just chemical bonding of growth factor on collagen surface, the general click chemistry based on the cycloaddition of azides and terminal alkynes would be sufficient. We chose SPAAC specifically due to its safety profile in living systems and potential for in vitro and in vivo applications in the presence of living cells. The problem of general click chemistry is that the reaction requires copper for catalysis, which is cytotoxic.28 Thus, copper-based click chemistry is hard to apply in vitro or in vivo environments. Therefore, we used copper-free click chemistry using SPAAC as an alternative chemical reaction to be utilized directly to the tissues. To the best of our knowledge, there is currently no in situ application of SPAAC to anchor exogenous biomolecules to tissue in vivo, though the reaction is promising because of its biocompatibility and cytocompatibility. Performing SPAAC in vivo requires the conjugation of azide and alkyne groups onto exogenous biomolecules and to endogenous biomolecules on tissue surfaces. We used NHS coupling chemistry to derivatize EGF and collagen-coated surfaces with azide and DBCO groups, respectively. The main advantages of the NHS ester reaction is that it is rapid and can take place at room temperature within a few minutes.29 In our case, NHS ester coupling was effective at



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introducing SPAAC reaction groups, because both collagen, which is the predominant structural protein in human tissue, and EGF which is wound healing promoter, both have primary amine groups. The chemicals of DBCO- and azide-containing NHS ester group were utilized for modification of the collagen surface and EGF, respectively. Assuming clinical application, the DBCO group can modify not only tissue surfaces but also other biomolecules; however, other molecules could be washed out since they are not fixed on the tissue surface. The EGF can be modified in a well-controlled environment before application, while the collagen conjugation can be carried out under controlled conditions as well, and in situ. Another advantage of our strategy is the cytocompatibility of chemical reaction. To eliminate every possibility of risk against harmful reagent including organic solvents such as dimethyl sulfoxide (DMSO), we used only water and PBS based solution in the whole process. Since the NHS ester is not soluble in aqueous solution, DMSO has been used in the past as a solvent for dissolving NHS ester reagents. Even though DMSO has been widely used in chemistry and biology as a solvent, its safety for clinical use is not clear. In this study, to avoid using DMSO, we used water soluble sulfo-NHS ester which is hydrophilic and maintains high reactivity with amine groups.9 DBCO-sulfo-NHS ester and azide-PEG-sulfo-NHS ester were applied, and both commercially available reagents were water soluble. Moreover, since the simple conjugation processes are progressed by solution mixing at 4 °C or room temperature, not only the reagent but also the reaction conditions are biocompatible. In the azide and DBCO conjugation to EGF, the maintenance of bioactivity is more important than the number of substitutes. EGF has two critical receptor binding residues at Arg41 and Leu47,20 which represent that conjugation of Azide-PEG group at the amines of N-terminal and Lys28 would give less impact on the bioactivity of EGF since they are far away from the critical


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receptor binding residues. On the other hand, the Lys48 is next to the Leu47, so it is expected that the conjugation of Azide-PEG group at Lys48 brings about steric hindrance to the EGF/EGF receptor binding. Unfortunately, despite the high nucleophilicity of the N-terminal amine, all the reactivities of amines were almost equal due to the high reactivity of NHS ester.20 A limitation of our study is that Azide-PEG-EGF retained some but not all of its bioactivity, which was confirmed by the EGF/EGFR binding assay (Figure 2b). The bioactive composition was 42.18 ± 7.08 %. We assumed that three primary amines have equal reactivity to NHS ester reaction and EGF loses activity when azide was conjugated to Lys48 in the mass spectrometry result (Figure 2a). EGFs that have one, two and three azide-PEG groups would be not active one-third, twothird and all, respectively. Based on the relative ratio provided by MALDI-TOF, roughly 46 % of Azide-PEG-EGF with unmodified EGF is active, which matches with bioactivity of EGF/EGFR binding assay. The selective modification by NHS ester to the N-terminal primary amine group would be necessary for maintaining bioactivity and efficiency after conjugation. We aim to control the conjugation of click moieties to improve bioactivity of growth factor after chemical modification in the future. Nevertheless, bioactive Azide-PEG-EGFs remains in the Azide-PEG modified EGF mixture, and was found to have trophic effects on cultured cells in chemically immobilized form. Interestingly, although the DBCO group has similar molecular weight to azide-PEG group, its hydrophobicity appears to have a negative effect on the bioactivity of EGF. Wang et al. mentioned that the modification with DBCO group can decrease ligand binding due to the great hydrophobicity.30 Here, the DBCO was used for collagen surface modification instead, because in our experience it does not impact the bioactivity of collagen, which is a much larger molecule with numerous cell adhesion domains.



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In this study, we applied various concentrations of reagents depending on the characterization methods to show effects of chemical immobilization. For surface characterizations including ellipsometry, XPS, SPR and ELISA, 0.1 mg/mL of sufficient DBCO-sulfo-NHS ester was applied to provide DBCO groups on the collagen surface, with unreacted reagents washed out. DBCO is relatively small compared to EGF and collagen, so it did not alter surface layer thickness, but produced a dramatic difference in surface chemical composition of carbon (Figure 4c). For in vitro cell experiments, the concentration was adjusted to 0.025 mg/mL due to observed cytotoxicity. We found 0.01 mg/mL of Azide-PEG-EGF was enough to see the difference compared collagen coating or physical adsorption for ellipsometry and XPS (Figure 3 and 4). For SPR measurements, at least 0.1 mg/mL of Azide-PEG-EGF was required to observe real-time variation with continuous fluid and short times (Figure 5). For ELISA experiments, the concentration was reduced to 0.001 mg/mL because higher concentrations exceeded the limitation of ELISA since the linear range of the kit was under about 80 pg/cm2. Also, the 0.01 mg/mL was found to be the concentration that provided significant differences on corneal epithelial cell behavior in culture (Figure 8). We anticipate that as this work progresses further iterations on the reaction conditions will be required to optimize the biological response to this process. Here, the chemically immobilized EGF in measurable condition was found to be 69.76 ± 0.94 pg/cm2 with 0.1 mg/mL of DBCO-sulfo-NHS ester, 0.01 mg/mL of Azide-PEG-EGF and 60 minutes of reaction time (Figure 6c), which can be seen to be small compared to other previous studies. For example, the range was about 18 – 163 ng/cm2 in the previous studies,31-33 therefore our result showed lower concentrations than this. However, there were vast differences in the reaction condition. First, the concentration of carbodiimide or carbonyldiimidazole which were


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utilized chemical linker was 5 – 30 mg/mL,31, 33 while 0.1 mg/mL of DBCO-sulfo-NHS ester was used for surface modification for SPAAC coupling. As we showed in Figure 6a, the concentration of chemical linker has a great influence to immobilized growth factor. A direct comparison among concentrations can be difficult because of the different types of chemical, but it is evident that higher concentration of chemical linker for immobilization is beneficial. Second, 10 – 50 times more EGF were applied than our procedure.31-33 It is estimated to be immobilized at least two-fold higher if we applied a ten-fold concentration of Azide-PEG-EGF based on concentrations the between 0.001 and 0.01 mg/mL in Figure 6b. However, as we mentioned above, the value at 0.01 mg/mL is in the saturated region of ELISA. Because the actual immobilized growth factor would be greater than the measured value, the substantive effect on increased concentration is likely to be considerably higher than our estimation. Third, the incubation time range was entirely different. Previous studies allowed to proceed for 24 and 48 hours,31-33 but this time frame is impractical from a clinical perspective. We demonstrated a relatively fast SPAAC reaction, with tethering occurring in as little as 10 minutes (Figure 6c). Although the reaction would not continue for more than 24 hours, it can be expected that more EGF will be immobilized from the increasing trend up to 60 minutes. As far as we can judge, our quantification result is reasonable compared other studies, and may be applicable to clinical use. The effects of EGF on cells established, and the distinctive influence of chemically immobilized EGF on the cell morphology and proliferation we found were consistent with reports by previous researchers.31,

34

The primary mechanism of promoting corneal epithelial

proliferation and migration by EGF is phosphorylation of EGFR by ligand binding,31, 34-35 which activates several signaling pathways such as mitogen-activated protein kinase (MAPK), extracellular signal–regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt



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pathways related to the cell migration and proliferation.34-35 Although the specific effects on these pathways is outside the scope of this paper, our results indicate that the coupled EGF exerts persistent biological effects over time while the effects of soluble or adsorbed EGF was transient with a rapid reduction of effects over time.34

◼ CONCLUSIONS Direct surface-coupling of growth factors on collagen surfaces was performed by SPAAC. Azide groups were conjugated to EGF via NHS ester chemistry, and the number of azide groups conjugated to EGF and bioactivity of modified EGFs were evaluated. By the introduction of DBCO groups to collagen surfaces and subsequent SPAAC reaction to azide-modified EGF, the biomolecule was chemically immobilized to the collagen surface successfully and confirmed by several surface analysis techniques. The chemically immobilized EGF showed robust bonding that maintained layer thickness after washing, while physically adsorbed EGF was readily washed away. SPAAC-mediated chemical bonding and its individual reactive elements were not cytotoxic and biocompatible within a defined range, which was evaluated by in vitro stromal cells viability assay with a model collagen coating. Chemically immobilized EGF promoted improved adhesion and proliferation of corneal epithelial cells compared to soluble EGF application. In summary, we have developed a novel approach for chemical immobilization of growth factors to collagen surfaces using copper-free click chemistry that is biocompatible, highly selective, and active in mild aqueous conditions without harmful organic solvents, and thus provides a robust method for biocompatibly modifying surfaces both in vitro and in vivo in the presence of living cells.


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◼ AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected] * E-mail address: [email protected]

Author Contributions † H.J.L. and G.M.F.-C. contributed equally to this work.

◼ ACKNOWLEDGMENT This work was supported by Stanford SPARK Translational Research Grant and the Byers Eye Institute at Stanford. Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-1542152.

◼ ABBREVIATIONS DBCO, dibenzocyclooctyne; DMSO, dimethyl sulfoxide; EDC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal–regulated kinase; MALDI-TOF, matrix-assisted laser desorption and ionization time of flight; MAPK, mitogen-activated protein kinase; NHS, Nhydroxysuccinimide; PEG, poly(ethylene glycol); PI3-K, phosphoinositide 3-kinase; RU, response unit; SPAAC, strain-promoted azide-alkyne cycloaddition; SPR, surface plasmon resonance; XPS, x-ray photoelectron spectroscopy.

◼ REFERENCES



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